表观遗传学修饰下的miR-193a/miR-199a-1在AML中协同抑制WT1蛋白的机制研究

The aberrant overexpression of the wilm's tumor-1(WT1) in AML plays an important role in pathogenesis of AML (Acute myeloid leukemia). However, the underlying mechanism is still unknown. Our previous data showed that miR-193a and miR-199a-1 synergistically repressed WT1 expression through binding coding sequence (CDS) and 3'-untranslated regions (3'UTR) of WT1 mRNA, respectively. More importantly, the expression levels of miR-193a and miR-199a-1 was downregulated in AML blast than normal controls due to the hypermethylation in promoter region. Thus, we speculate that the silencing of miR-193a and miR-199a-1 by DNA hypermethylation may account for the high expression of WT1 in AML. To further validate the negative regulation mechanism of WT1 gene by miR-193a/199a-1, we will carry out luciferase reporter assay, clinical data analysis of AML patients, and animal model. Furthermore, standard methylation-specific polymerase chain reaction and sequencing will be used to compare the status of miR-193a/199a-1 methylation in AML blasts and healthy individuals. Our findings demonstrate that miRNAs may serve as targets for leukemia prevention and therapy, which has a strong theoretical significance and application prospects for the AML patients with high expression of WT1.

Wilm’s tumor-1(WT1)基因在绝大部分AML细胞中高表达，然而高表达的机制一直不是很清楚。我们前期工作发现，miR-193a和miR-199a-1分别通过结合WT1基因编码区和3'端非编码区协同抑制WT1表达。而miR-193a/199a-1因为启动子高甲基化而在白血病细胞中表达沉默，从而失去了对WT1的抑制作用，这可能是WT1基因在白血病细胞中高表达的重要原因。本课题拟进一步通过荧光素酶报告基因实验、白血病病人临床资料分析及动物实验全方位验证miR-193a/199a-1负性调控WT1机制。通过甲基化特异PCR验证miR-193a/199a-1在白血病细胞及正常对照细胞中的甲基化水平。本研究初步探讨了以miRNA为靶点治疗高表达WT1白血病的可能性，为高表达WT1的白血病治疗提供新思路和新策略，具有很强的理论意义和应用前景。

Wilm’s-tumor-1 (WT1)在急性髓细胞白血病细胞中的高表达导致白血病细胞的恶性增殖，是造成白血病患者生存期减少的重要原因。然而，WT1在白血病细胞中表达增高的机制尚不清楚。MicroRNA（miRNA）是一类真核生物高度保守的内源性小调节RNA，通过结合目标基因的非编码区或者编码区，抑制目标基因的表达。我们发现两种微小RNA，miR-193a和miR-600通过分别结合WT1基因编码区和3'非编码区，协同抑制WT1基因表达。然而，miR-193a和miR-600在白血病细胞中表达降低，失去对WT1的抑制能力，导致WT1表达增高。miR-193a在白血病细胞中存在高度甲基化，高甲基化导致miR-193a在白血病细胞中低表达。WT1结合miR-600启动子，抑制miR-600表达。因此，miR-193a/600和WT1形成负性调控网络。应用去甲基化药物地西他滨(5'-azacytidine, AZA)逆转miR-193a启动子甲基化，恢复miR-193a表达，从而阻断miR-193a/600-WT1调控网络，抑制WT1表达。在白血病细胞中过表达miR-193a/600，抑制增殖，促进凋亡和抑制集落形成。我们进一步通过裸鼠成瘤实验，发现miR-193a/miR-600明显抑制K562瘤体的大小和重量。通过白血病细胞移植实验发现miR-193a/miR-600明显减少人THP1白血病细胞在免疫缺陷小鼠(NSG)外周血的侵袭，并且显著延长NSG小鼠总生存期。我们的体内实验结果表明恢复miR-193a/miR-600表达具有显著的抗白血病效果，并延长小鼠生存期，提示在临床白血病患者中恢复miR-193a/miR-600表达，并联合常规化疗方案，可能有助于提高临床治疗效果。本课题首次通过miRNA途径探讨WT1在白血病细胞中高表达的机制，发现了两种能够协同抑制WT1的miRNAs，为WT1高表达的白血病患者治疗提供新思路和新策略，具有很强的理论意义和应用前景。